Titanium Dioxide (E171 Grade) and the Search For Replacement Opacifiers and Colorants: Supplier Readiness Survey, Case Studies and Regulatory Perspective.

Titanium dioxide (TiO2) is used primarily as an opacifier in solid dosage forms and is present in the majority of tablet and capsule dosage forms on the market. The IQ* TiO2 Working Group has previously shown that titanium dioxide has unique properties which are necessary for its function in these formulations and noted that, as the potential replacements lack the semi-conductor properties, high refractive index and whiteness of E171, it might be hard to replicate these properties with alternative materials. In this paper we detail the results of readiness surveys and practical assessments that have been conducted with alternative materials by IQ member companies. A range of technical challenges and regulatory hurdles were identified which mean that, in the short term, it may be difficult to replace titanium dioxide with the currently available alternative materials while readily achieving the same drug product quality attributes, especially for some of the marketed formulations that titanium dioxide is currently used for. We note the higher technical complexity, due to the variability, color fading and identified scale up risk, of E171 free film coatings and the likely impact on development costs and timelines. We also highlight several regulatory hurdles that would have to be overcome if a titanium dioxide replacement was required for some markets but was not mandated by others.

Excitation-transcription coupling, neuronal gene expression and synaptic plasticity.

Excitation-transcription coupling (E-TC) links synaptic and cellular activity to nuclear gene transcription. It is generally accepted that E-TC makes a crucial contribution to learning and memory through its role in underpinning long-lasting synaptic enhancement in late-phase long-term potentiation and has more recently been linked to late-phase long-term depression: both processes require de novo gene transcription, mRNA translation and protein synthesis. E-TC begins with the activation of glutamate-gated N-methyl-D-aspartate-type receptors and voltage-gated L-type Ca2+ channels at the membrane and culminates in the activation of transcription factors in the nucleus. These receptors and ion channels mediate E-TC through mechanisms that include long-range signalling from the synapse to the nucleus and local interactions within dendritic spines, among other possibilities. Growing experimental evidence links these E-TC mechanisms to late-phase long-term potentiation and learning and memory. These advances in our understanding of the molecular mechanisms of E-TC mean that future efforts can focus on understanding its mesoscale functions and how it regulates neuronal network activity and behaviour in physiological and pathological conditions.

Opto4E-BP, an optogenetic tool for inducible, reversible, and cell type-specific inhibition of translation initiation.

The protein kinase mechanistic target of rapamycin complex 1 (mTORC1) is one of the primary triggers for initiating cap-dependent translation. Amongst its functions, mTORC1 phosphorylates eIF4E-binding proteins (4E-BPs), which prevents them from binding to eIF4E and thereby enables translation initiation. mTORC1 signaling is required for multiple forms of protein synthesis-dependent synaptic plasticity and various forms of long-term memory (LTM), including associative threat memory. However, the approaches used thus far to target mTORC1 and its effectors, such as pharmacological inhibitors or genetic knockouts, lack fine spatial and temporal control. The development of a conditional and inducible eIF4E knockdown mouse line partially solved the issue of spatial control, but still lacked optimal temporal control to study memory consolidation. Here, we have designed a novel optogenetic tool (Opto4E-BP) for cell type-specific, light-dependent regulation of eIF4E in the brain. We show that light-activation of Opto4E-BP decreases protein synthesis in HEK cells and primary mouse neurons. In situ , light-activation of Opto4E-BP in excitatory neurons decreased protein synthesis in acute amygdala slices. Finally, light activation of Opto4E-BP in principal excitatory neurons in the lateral amygdala (LA) of mice after training blocked the consolidation of LTM. The development of this novel optogenetic tool to modulate eIF4E-dependent translation with spatiotemporal precision will permit future studies to unravel the complex relationship between protein synthesis and the consolidation of LTM.

A high-throughput screening to identify small molecules that suppress huntingtin promoter activity or activate huntingtin-antisense promoter activity.

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of huntingtin (HTT). While there are currently no disease-modifying treatments for HD, recent efforts have focused on the development of nucleotide-based therapeutics to lower HTT expression. As an alternative to siRNA or oligonucleotide methods, we hypothesized that suppression of HTT expression might be accomplished by small molecules that either (1) directly decrease HTT expression by suppressing HTT promoter activity or (2) indirectly decrease HTT expression by increasing the promoter activity of HTT-AS, the gene antisense to HTT that appears to inhibit expression of HTT. We developed and employed a high-throughput screen for modifiers of HTT and HTT-AS promoter activity using luminescent reporter HEK293 cells; of the 52,041 compounds tested, we identified 898 replicable hits. We used a rigorous stepwise approach to assess compound toxicity and the capacity of the compounds to specifically lower huntingtin protein in 5 different cell lines, including HEK293 cells, HD lymphoblastoid cells, mouse primary neurons, HD iPSCs differentiated into cortical-like neurons, and HD hESCs. We found no compounds which were able to lower huntingtin without lowering cell viability in all assays, though the potential efficacy of a few compounds at non-toxic doses could not be excluded. Our results suggest that more specific targets may facilitate a small molecule approach to HTT suppression.

Excitatory and inhibitory synapse reorganization immediately after critical sensory experience in a vocal learner.

Excitatory and inhibitory synapses are the brain's most abundant synapse types. However, little is known about their formation during critical periods of motor skill learning, when sensory experience defines a motor target that animals strive to imitate. In songbirds, we find that exposure to tutor song leads to elimination of excitatory synapses in HVC (used here as a proper name), a key song generating brain area. A similar pruning is associated with song maturation, because juvenile birds have fewer excitatory synapses, the better their song imitations. In contrast, tutoring is associated with rapid insertion of inhibitory synapses, but the tutoring-induced structural imbalance between excitation and inhibition is eliminated during subsequent song maturation. Our work suggests that sensory exposure triggers the developmental onset of goal-specific motor circuits by increasing the relative strength of inhibition and it suggests a synapse-elimination model of song memorization.